The present invention is in the area of improved procaryotic expression systems and, in particular, a Coryneform host system for the expression and excretion of gene products.
Coryneform bacteria are a taxonomically ill-defined group of Gram positive bacteria originally related by unique morphological features. These microorganisms occupy a wide variety of ecological niches and display an even broader array of interesting and useful properties. With the advent of systematic chemical analysis, there is considerable evidence indicating that the genus Corynebacterium is closely related to Mycobacterium and Norcardia. Included in the genus Corynebacterium are medically important species such as C. dipthereiae, animal pathogens such as C. renale, plant pathogens and diverse saprophytic, aerobic coryneform bacteria. The saprophytic coryneform bacteria are widely distributed in nature and include not only Corynebacterium species but also other bacteria including Arthrobacter, Brevibacterium, Cellulomonas, Microbacterium and Curtobacterium. The coryneform group thus represents an important source of enzymes, primary metabolites, and genetic material.
When cloning heterologous proteins for purification, it is often desirable to have the gene product hyperproduced and/or secreted by the host cells. The major advantages of secretion over intracellular accumulation of recombinant proteins are an increase in yield and the facilitation of product purification. Translocation of proteins into or through membranes is an essential feature of prokaryotic and eukaryotic cells. Proteins that are partially or fully integrated into membranes, proteins that are associated or covalently bound to cell walls, or proteins that are secreted, must cross the cytoplasmic membrane.
Although initial investigations on protein export have beer carried out with eukaryotic systems, there is an increasing interest in the mechanism and genetics of bacterial protein export. Benson et al., Cell 32, 1325-1335 (1985); D. Oliver, Ann.Rev.Microbirl. 39,615-648 (1985); Randall and Hardy, Microbiol.Rev. 48, 290-298 (1984); and Pugsley and Schwartz, FEMS Microbiol.Rev. 48,290-298 (1985), have recently reviewed this area. The Gram negative E. coli is the best-studied species among the prokaryotes. The most advanced experimental techniques have been tailored especially to fit the E. coli system. Despite the fact that the Gram positive cell well has a simpler structure than its Gram negative counterpart, that Gram positive organisms are often very efficient in secreting proteins to the culture medium as compared with Gram negative organisms which normally cannot transport proteins beyond the outer membrane of their cell envelope, and that a vast number of extracellular proteins of Gram positive bacteria have been isolated and examined, including most bacterial enzymes of commercial importance, the use of these organisms for basic investigations of protein export has been limited.
Most exported proteins, contrary to the majority of proteins localized in the cytoplasmic membrane, are synthesized as precursors with an N-terminal peptide extension (signal peptide) that is cleaved off in the course of translocation. Many of the bacterial and eukaryotic signal sequences that have been studied share striking structural similarities and are in fact interchangeable, as reported by several investigators. For example, the E. coli leader peptidase precisely recognizes and cleaves eukaryotic precursors.
Protein fusion experiments have demonstrated that a signal sequence alone is generally insufficient for the proper export of proteins. Several other types of targeting signals in addition to signal peptides have been identified. The most complex situation is found in eukaryotic cells where proteins must be directed to different subcellular compartments: endoplasmic reticulum, mitochondria, or chloroplasts. Additional information in the body of the mature protein may also be necessary. For example, posttranslational modification may contribute to the final localization of a protein, as seen with Gram negative lipoproteins and Gram positive lipopenicillinases.
Unfortunately, at this time, a good Gram positive cloning host has not been identified. The classic Gram positive cloning host, B. subtilis, secretes extracellular proteases which attack heterologous proteins expressed in this organism, as reported by Ulmanen et al., J.Bacteriol. 162,176-182 (1985) and Doi et al., Trends in Biotech. 232-235 (1986). Consequently, there is a clear need for alternative Gram positive host organisms.
Protein secretion by coryneform bacteria has not been investigated, other than the secretion of diphtheria toxin by the pathogenic C. diptheriae upon infection with certain lysogenic tox.sup.+ phages, reported by Pappenheimer, Ann.Rev.Biochem. 46,69-94 (1977) and NeviIle and Hudson, Ann.Rev.Biochem. 55,195-224 (1986). Even reports of the cloning in Corynebacterium hosts of the genes for two proteins which are normally exported in their native hosts, beta-lactamase from E. coli and alpha-amylase from Bacillus amyloliquefaciens, do not disclose whether or not these heterologous proteins were secreted.
It is therefore an object of the present invention to provide a Gram positive bacterial expression and secretion system.
It is another object of the present invention to characterize gene expression (replication, conjugal transfer and plasmid biology), in the Gram positive bacterial expression system.
It is yet another object of the present invention to further elucidate the genomic organization and structure of the Gram positive host, including the isolation and characterization of high efficiency and regulatable promoters.